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• W2165087479 abstract "Atherosclerosis is a state of heightened oxidative stress. Oxidized LDL is present in atherosclerotic lesions and used as marker for coronary artery disease, although in human lesions lipids associated with HDL are as oxidized as those of LDL. Here we investigated specific changes occurring to apolipoprotein A-I (apoA-I) and apoA-II, as isolated HDL and human plasma undergo mild, chemically induced oxidation, or autoxidation. During such oxidation, Met residues in apoA-I and apoA-II become selectively and consecutively oxidized to their respective Met sulfoxide (MetO) forms that can be separated by HPLC. Placing plasma at −20°C prevents autoxidation, whereas metal chelators and butylated hydroxytoluene offer partial protection. Independent of the oxidation conditions, apoA-I and apoA-II (dimer) with two MetO residues accumulate as relatively stable oxidation products. Compared to controls, serum samples from subjects with the endothelial cell nitric oxide synthase a/b genotype that is associated with increased coronary artery disease contain increased concentrations of apoA-I with two MetO residues.Our results show that during the early stages, oxidation of HDL gives rise to specifically oxidized forms of apoA-I and apoA-II, some of which may be useful markers of in vivo HDL oxidation, and hence potentially atherosclerosis. Atherosclerosis is a state of heightened oxidative stress. Oxidized LDL is present in atherosclerotic lesions and used as marker for coronary artery disease, although in human lesions lipids associated with HDL are as oxidized as those of LDL. Here we investigated specific changes occurring to apolipoprotein A-I (apoA-I) and apoA-II, as isolated HDL and human plasma undergo mild, chemically induced oxidation, or autoxidation. During such oxidation, Met residues in apoA-I and apoA-II become selectively and consecutively oxidized to their respective Met sulfoxide (MetO) forms that can be separated by HPLC. Placing plasma at −20°C prevents autoxidation, whereas metal chelators and butylated hydroxytoluene offer partial protection. Independent of the oxidation conditions, apoA-I and apoA-II (dimer) with two MetO residues accumulate as relatively stable oxidation products. Compared to controls, serum samples from subjects with the endothelial cell nitric oxide synthase a/b genotype that is associated with increased coronary artery disease contain increased concentrations of apoA-I with two MetO residues. Our results show that during the early stages, oxidation of HDL gives rise to specifically oxidized forms of apoA-I and apoA-II, some of which may be useful markers of in vivo HDL oxidation, and hence potentially atherosclerosis. The oxidation of LDL is widely thought to be critical to atherogenesis (1Steinberg D. Parthasarathy S. Carew T.E. Khoo J.C. Witztum J.L. Beyond cholesterol: Modifications of low-density lipoprotein that increase its atherogenicity.N. Engl. J. Med. 1989; 320: 915-924Google Scholar). Oxidation of the LDL-associated antioxidants ubiquinol-10 (2Stocker R. Bowry V.W. Frei B. Ubiquinol-10 protects human low density lipoprotein more efficiently against lipid peroxidation than does α-tocopherol.Proc. Natl. Acad. Sci. USA. 1991; 88: 1646-1650Google Scholar) and α-tocopherol (3Esterbauer H. Jürgens G. Quehenberger O. Koller E. Autoxidation of human low density lipoprotein: loss of polyunsaturated fatty acids and vitamin E and generation of aldehydes.J. Lipid Res. 1987; 28: 495-509Google Scholar) represents the initial step in this process, concomitant with formation of lipid hydroperoxides (2Stocker R. Bowry V.W. Frei B. Ubiquinol-10 protects human low density lipoprotein more efficiently against lipid peroxidation than does α-tocopherol.Proc. Natl. Acad. Sci. USA. 1991; 88: 1646-1650Google Scholar, 4Upston J.M. Terentis A.C. Stocker R. Tocopherol-mediated peroxidation (TMP) of lipoproteins: implications for vitamin E as a potential antiatherogenic supplement.FASEB J. 1999; 13: 977-994Google Scholar) that in turn may then lead to oxidation of apolipoprotein B-100 (apoB-100) (5Steinbrecher U.P. Lougheed M. Kwan W-C. Dirks M. Recognition of oxidized low density lipoprotein by the scavenger receptor of macrophages results from derivatization of apolipoprotein B by products of fatty acid peroxidation.J. Biol. Chem. 1989; 264: 15216-15223Google Scholar). Oxidized LDL (oxLDL) is present in human atherosclerotic lesions (6Ylä-Herttuala S. Palinski W. Rosenfeld M.E. Parthasarathy S. Carew T.E. Butler S. Witztum J.L. Steinberg D. Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man.J. Clin. Invest. 1989; 84: 1086-1095Google Scholar), and its detection in circulation by immunological assays is used as a surrogate for, or marker of, atherosclerotic disease (7Holvoet P. Perez G. Zhao Z. Brouwers E. Bernar H. Collen D. Malondialdehyde-modified low density lipoproteins in patients with atherosclerotic disease.J. Clin. Invest. 1995; 95: 2611-2619Google Scholar, 8Ehara S. Ueda M. Naruko T. Haze K. Itoh A. Otsuka M. Komatsu R. Matsuo T. Itabe H. Takano T. Tsukamoto Y. Yoshiyama M. Takeuchi K. Yoshikawa J. Becker A.E. Elevated levels of oxidized low density lipoprotein show a positive relationship with the severity of acute coronary syndromes.Circulation. 2001; 103: 1955-1960Google Scholar). However, the term oxLDL refers to a mixture of modified lipoproteins that remain chemically uncharacterized (9Steinberg D. Oxidized low density lipoprotein–an extreme example of lipoprotein heterogeneity.Isr. J. Med. Sci. 1996; 32: 469-472Google Scholar). This limits the use of measurement of oxLDL as a diagnostic for cardiovascular disease(s). In addition, there are several caveats concerning the accuracy of these measurements, including the extent of LDL oxidation, reproducibility of the reference oxLDL, and a better characterization of the epitope recognized by the antibody used (10Tsimikas S. Witztum J.L. Measuring circulating oxidized low-density lipoprotein to evaluate coronary risk.Circulation. 2001; 103: 1930-1932Google Scholar). HDLs are also subject to oxidative modification in vivo, as HDL lipids are at least as susceptible to oxidation as those of LDL (11Bowry V.W. Stanley K.K. Stocker R. High density lipoprotein is the major carrier of lipid hydroperoxides in fasted human plasma.Proc. Natl. Acad. Sci. USA. 1992; 89: 10316-10320Google Scholar). Also, lipids in HDL and LDL isolated from human atherosclerotic lesions are oxidized to a comparable extent (12Niu X. Zammit V. Upston J.M. Dean R.T. Stocker R. Co-existence of oxidized lipids and α-tocopherol in all lipoprotein fractions isolated from advanced human atherosclerotic plaques.Arterioscler. Thromb. Vasc. Biol. 1999; 19: 1708-1718Google Scholar) that increases with increasing severity of disease (13Upston J.M. Niu X. Brown A.J. Mashima R. Wang H. Senthilmohan R. Kettle A.J. Dean R.T. Stocker R. Disease stage-dependent accumulation of lipid and protein oxidation products in human atherosclerosis.Am. J. Pathol. 2002; 160: 701-710Google Scholar). These lesion lipoproteins retain normal concentrations of α-tocopherol (12Niu X. Zammit V. Upston J.M. Dean R.T. Stocker R. Co-existence of oxidized lipids and α-tocopherol in all lipoprotein fractions isolated from advanced human atherosclerotic plaques.Arterioscler. Thromb. Vasc. Biol. 1999; 19: 1708-1718Google Scholar, 14Suarna C. Dean R.T. May J. Stocker R. Human atherosclerotic plaque contains both oxidized lipids and relatively large amounts of α-tocopherol and ascorbate.Arterioscler. Thromb. Vasc. Biol. 1995; 15: 1616-1624Google Scholar, 15Upston J.M. Terentis A.C. Morris K. Keaney Jr., J.F. Stocker R. Oxidized lipid accumulates in the presence of a-tocopherol in atherosclerosis.Biochem. J. 2002; 363: 753-760Google Scholar, 16Terentis A.C. Thomas S.R. Burr J.A. Liebler D.C. Stocker R. Vitamin E oxidation in human atherosclerotic lesions.Circ. Res. 2002; 90: 333-339Google Scholar) and most of their major lipid oxidation products, i.e., cholesterylester hydro(pero)xides, accumulate in the presence of the vitamin (15Upston J.M. Terentis A.C. Morris K. Keaney Jr., J.F. Stocker R. Oxidized lipid accumulates in the presence of a-tocopherol in atherosclerosis.Biochem. J. 2002; 363: 753-760Google Scholar). These findings suggest that oxidized lesion lipoproteins, including oxHDL, that could be present in blood of subjects with atherosclerotic disease, include early stage, oxidized lipoproteins. Several considerations favor the potential use of oxHDLs or their component(s) over oxLDL as a potential marker of atherosclerotic disease. First, being substantially smaller and interacting less strongly with extracellular proteoglycans, vessel wall HDL is expected to re-enter the circulation more readily than LDL (17Borén J. Olin K. Lee I. Chait A. Wight T.N. Innerarity T.L. Identification of the principal proteoglycan-binding site in LDL. A single-point mutation in apo-B100 severely affects proteoglycan interaction without affecting LDL receptor binding.J. Clin. Invest. 1998; 101: 2658-2664Google Scholar). Second, whereas apoB-100 does not dissociate from LDL, HDL's apolipoproteins dissociate readily and limited oxidation enhances this process in the case of apoA-I (18Panzenböck U. Kritharides L. Raftery M. Rye K.A. Stocker R. Oxidation of methionine residues to methionine sulfoxides does not decrease potential anti-atherogenic properties of apolipoprotein A-I.J. Biol. Chem. 2000; 275: 19536-19544Google Scholar), further increasing the likelihood of its existence in circulation. Third, given their physical properties and smaller molecular size, oxidized forms of apoA-I and apoA-II are simpler to work with and to chemically characterize than apoB-100. Compared with LDL, relatively little is known about how HDLs and their components become oxidized. Previous in vitro studies utilized different oxidants, including H2O2 (19Anantharamaiah G.M. Hughes T.A. Iqbal M. Gawish A. Neame P.J. Medley M.F. Segrest J.P. Effect of oxidation on the properties of apolipoproteins A-I and A-II.J. Lipid Res. 1988; 29: 309-318Google Scholar, 20von Eckardstein A. Walter M. Holz H. Benninghoven A. Assmann G. Site-specific methionine sulfoxide formation is the structural basis of chromatographic heterogeneity of apolipoproteins A-I, C–II, and C–III.J. Lipid Res. 1991; 32: 1465-1476Google Scholar), myeloperoxidase-derived oxidants (21Francis G.A. Mendez A.J. Bierman E.L. Heinecke J.W. Oxidative tyrosylation of high density lipoprotein by peroxidase enhances cholesterol removal from cultured fibroblasts and macrophage foam cells.Proc. Natl. Acad. Sci. USA. 1993; 90: 6631-6635Google Scholar, 22Heinecke J.W. Li W. Francis G.A. Goldstein J.A. Tyrosyl radical generated by myeloperoxidase catalyzes the oxidative cross-linking of proteins.J. Clin. Invest. 1993; 91: 2866-2872Google Scholar, 23Panzenboeck U. Raitmayer S. Reicher H. Lindner H. Glatter O. Malle E. Sattler W. Effects of reagent and enzymatically generated hypochlorite on physicochemical and metabolic properties of high density lipoproteins.J. Biol. Chem. 1997; 272: 29711-29720Google Scholar, 24Bergt C. Oettl K. Keller W. Andreae F. Leis H.J. Malle E. Sattler W. Reagent or myeloperoxidase-generated hypochlorite affects discrete regions in lipid-free and lipid-associated human apolipoprotein A-I.Biochem. J. 2000; 346: 345-354Google Scholar), lipid hydroperoxides (25Sattler W. Christison J.K. Stocker R. Cholesterylester hydroperoxide reducing activity associated with isolated high- and low-density lipoproteins.Free Radic. Biol. Med. 1995; 18: 421-429Google Scholar, 26Garner B. Witting P.K. Waldeck A.R. Christison J.K. Raftery M. Stocker R. Oxidation of high density lipoproteins. I. Formation of methionine sulfoxide in apolipoproteins AI and AII is an early event that correlates with lipid peroxidation and can be enhanced by α-tocopherol.J. Biol. Chem. 1998; 273: 6080-6087Google Scholar, 27Mashima R. Yamamoto Y. Yoshimura S. Reduction of phosphatidylcholine hydroperoxide by apolipoprotein A-I: purification of the hydroperoxide-reducing proteins from human blood plasma.J. Lipid Res. 1998; 39: 1133-1140Google Scholar), and peroxyl radicals (26Garner B. Witting P.K. Waldeck A.R. Christison J.K. Raftery M. Stocker R. Oxidation of high density lipoproteins. I. Formation of methionine sulfoxide in apolipoproteins AI and AII is an early event that correlates with lipid peroxidation and can be enhanced by α-tocopherol.J. Biol. Chem. 1998; 273: 6080-6087Google Scholar) and Cu2+ (26Garner B. Witting P.K. Waldeck A.R. Christison J.K. Raftery M. Stocker R. Oxidation of high density lipoproteins. I. Formation of methionine sulfoxide in apolipoproteins AI and AII is an early event that correlates with lipid peroxidation and can be enhanced by α-tocopherol.J. Biol. Chem. 1998; 273: 6080-6087Google Scholar) that modify apoA-I and apo-II in different ways and to varying extent. In the case of apoA-I, HDL’s major apolipoprotein, a common feature of mild oxidation is that Met residues become oxidized to methionine sulfoxides (MetOs) (19Anantharamaiah G.M. Hughes T.A. Iqbal M. Gawish A. Neame P.J. Medley M.F. Segrest J.P. Effect of oxidation on the properties of apolipoproteins A-I and A-II.J. Lipid Res. 1988; 29: 309-318Google Scholar, 20von Eckardstein A. Walter M. Holz H. Benninghoven A. Assmann G. Site-specific methionine sulfoxide formation is the structural basis of chromatographic heterogeneity of apolipoproteins A-I, C–II, and C–III.J. Lipid Res. 1991; 32: 1465-1476Google Scholar, 24Bergt C. Oettl K. Keller W. Andreae F. Leis H.J. Malle E. Sattler W. Reagent or myeloperoxidase-generated hypochlorite affects discrete regions in lipid-free and lipid-associated human apolipoprotein A-I.Biochem. J. 2000; 346: 345-354Google Scholar, 26Garner B. Witting P.K. Waldeck A.R. Christison J.K. Raftery M. Stocker R. Oxidation of high density lipoproteins. I. Formation of methionine sulfoxide in apolipoproteins AI and AII is an early event that correlates with lipid peroxidation and can be enhanced by α-tocopherol.J. Biol. Chem. 1998; 273: 6080-6087Google Scholar, 28Garner B. Waldeck A.R. Witting P.K. Rye K-A. Stocker R. Oxidation of high density lipoproteins. II. Evidence for direct reduction of HDL lipid hydroperoxides by methionine residues of apolipoproteins AI and AII.J. Biol. Chem. 1998; 273: 6088-6095Google Scholar). Lipid hydroperoxides formed during HDL oxidation convert Met112 and Met86 of apoA-I to MetO (18Panzenböck U. Kritharides L. Raftery M. Rye K.A. Stocker R. Oxidation of methionine residues to methionine sulfoxides does not decrease potential anti-atherogenic properties of apolipoprotein A-I.J. Biol. Chem. 2000; 275: 19536-19544Google Scholar, 26Garner B. Witting P.K. Waldeck A.R. Christison J.K. Raftery M. Stocker R. Oxidation of high density lipoproteins. I. Formation of methionine sulfoxide in apolipoproteins AI and AII is an early event that correlates with lipid peroxidation and can be enhanced by α-tocopherol.J. Biol. Chem. 1998; 273: 6080-6087Google Scholar, 28Garner B. Waldeck A.R. Witting P.K. Rye K-A. Stocker R. Oxidation of high density lipoproteins. II. Evidence for direct reduction of HDL lipid hydroperoxides by methionine residues of apolipoproteins AI and AII.J. Biol. Chem. 1998; 273: 6088-6095Google Scholar). However, it remains unclear whether Met oxidation in HDL-associated apoA-I occurs in a stepwise or “none or two” manner, and how oxidation of HDL’s second most abundant apolipoprotein, apoA-II occurs. We therefore investigated the identity and rate of accumulation of oxidized apoA-I versus apoA-II formed in fresh human plasma undergoing autoxidation or exposed to chemically controlled oxidation by alkyl peroxyl radicals. All chemicals were obtained from Sigma (Australia) unless specified otherwise. For autoxidation experiments, freshly isolated lithium heparin plasma was incubated at 37°C under air for the indicated period of time, then snap frozen at −80°C to prevent further oxidation before analyses. Chemically controlled oxidation was achieved by the addition of 2,2′-azo-bis(2-amidinopropane) dihydrochloride (AAPH) (Wako, Japan), a generator of aqueous peroxyl radicals, prior to incubation of the plasma samples at 37°C. Where indicated, diethylenetriamine pentaacetic acid (DTPA) or butylated hydroxy toluene (BHT) was also added prior to incubation. HDL was isolated from plasma by density gradient ultracentrifugation using a Ultima-X Benchtop Centrifuge equipped with a TL100.4 rotor (Beckman Instruments) centrifuged for 3 h at 15°C and 100,000 rpm (29Sattler W. Mohr D. Stocker R. Rapid isolation of lipoproteins and assessment of their peroxidation by HPLC postcolumn chemiluminescence.Methods Enzymol. 1994; 233: 469-489Google Scholar). The resulting HDL (comprising both HDL2 and HDL3) was aspirated and gel-filtered (PD-10 column, Pharmacia, Sweden) to remove low molecular weight compounds using 10 mM phospate buffer, pH 7.4, containing 100 mM DTPA. Cord blood samples were collected post-partum after normal full-term pregnancy and uncomplicated delivery from subjects recruited consecutively within the study period. We chose cord blood samples as they are less likely affected by environmental factors such as dietary vitamin supplements, as observed frequently in adult populations. The endothelial nitric oxide synthase (eNOS) intron 4 a/b polymorphism was determined from the cord blood DNA as described previously (30Wang X.L. Sim A.S. Wang M.X. Murrell G.A. Trudinger B. Wang J. Genotype dependent and cigarette specific effects on endothelial nitric oxide synthase gene expression and enzyme activity.FEBS Lett. 2000; 471: 45-50Google Scholar). Compared with the b/b genotype, carriers of the eNOSa allele who smoke have an increased risk for coronary artery stenosis and myocardial infarction (31Wang X.L. Sim A.S. Badenhop R.F. McCredie R.M. Wilcken D.E. A smoking-dependent risk of coronary artery disease associated with a polymorphism of the endothelial nitric oxide synthase gene.Nat. Med. 1996; 2: 41-45Google Scholar). Smoking status of the mothers during pregnancy was also documented. Blood was centrifuged within 10 min of collection and serum samples stored at −70°C within a further 10 min. HDL was then isolated and analyzed immediately for oxidized apolipoproteins by the HPLC method described below. Aliquots (typically 100 μl) of freshly isolated HDL (diluted 1:1, v/v, in HPLC-grade water) were applied to a 5 μm, 25 × 0.46 cm reverse phase C18 column (Vydac). The column was eluted at 0.5 ml/min and 50°C with an acetonitrile/water gradient containing 0.1% (v/v) TFA (Pierce) monitored at 214 nm. Following initial equilibration at 25% acetonitrile, the concentration was increased linearly to 45% over 5 min, and then to 55% over an additional 32 min. The acetonitrile content was then increased rapidly to 95% for 10 min and finally decreased to 25% for column re-equilibration. Oxidized and non-oxHDL samples were subjected to HPLC, and the protein fractions collected, pooled, and lyophilized. Mass spectra were acquired using a single quadrupole mass spectrometer equipped with an electrospray ionization source (Platform, VG-Fisons Instruments). Samples (50 pmol, 10 μl) were injected into a moving solvent (10 μl/min) of acetonitrile-water (1:1, v/v) coupled directly to the ionization source. MALDI/peptide mass finger printing spectra of lyophilized proteins from HPLC were determined after digestion with endoprotease AspN or trypsin (∼100 ng) in NH4HCO3 (25 μl, 20 mM, pH 8). After 14 h at 37°C, digests (1 μl) were analyzed directly after addition of matrix (DHB, 1 μl, 10 mg/ml) by MADLI over a mass range of m/z 500 to 7,000. Approximately 100 spectra were acquired in reflectron mode (Voyager STR, Perseptive Biosystems, Framingham, MA) with an accelerating voltage of 25,000 V. An extraction delay of 175 ns and spectra were calibrated externally using angiotensin I and insulin (ox) B chain. Peptides were identified by comparison with theoretically determined peptide masses. To assess the oxidation of HDL apolipoproteins in more detail, we first improved the HPLC method used previously (26Garner B. Witting P.K. Waldeck A.R. Christison J.K. Raftery M. Stocker R. Oxidation of high density lipoproteins. I. Formation of methionine sulfoxide in apolipoproteins AI and AII is an early event that correlates with lipid peroxidation and can be enhanced by α-tocopherol.J. Biol. Chem. 1998; 273: 6080-6087Google Scholar), utilizing an initial acetonitrile concentration of 25% (v/v) that was increased to 45% over 5 min, followed by a slower gradient to 65% of 30 min. Addition of this initial rapid increase in acetonitrile concentration improved the separation of HDL-associated proteins (data not shown). In native HDL, apoA-I and apoA-II were the two major proteins detected (Fig. 1A). ApoA-I eluted as a single species with a mass of 28081 Da as determined by electrospray mass spectrometry (predicted mass 28,078.7 Da). ApoA-II, which exists as a disulfide-linked homodimer, eluted as three distinct species with masses of 17,382, 17,255, and 17,123 Da, respectively. The largest of the three species corresponded to apoA-II homodimer with both N-terminal glutamine residues cyclized (predicted mass 17,381.8 Da), as described previously (32Brewer H.B.J. Lux S.E. Ronan R. John K.M. Amino acid sequence of human apoLp-Gln-II (apoA-II), an apolipoprotein isolated from the high-density lipoprotein complex.Proc. Natl. Acad. Sci. USA. 1972; 69: 1304-1308Google Scholar). Based on the mass changes, the two smaller species were assigned to apoA-II with one or both of the C-terminal glutamine residues removed (predicted masses 17,253.6 and 17,125.4 Da, respectively). We then exposed HDL to mild controlled oxidation by exposure to the free radical generator AAPH (1 mM, 37°C). This resulted in the time-dependent consumption of endogenous α-tocopherol during which phospholipid- and cholesterylester hydroperoxides (11Bowry V.W. Stanley K.K. Stocker R. High density lipoprotein is the major carrier of lipid hydroperoxides in fasted human plasma.Proc. Natl. Acad. Sci. USA. 1992; 89: 10316-10320Google Scholar, 26Garner B. Witting P.K. Waldeck A.R. Christison J.K. Raftery M. Stocker R. Oxidation of high density lipoproteins. I. Formation of methionine sulfoxide in apolipoproteins AI and AII is an early event that correlates with lipid peroxidation and can be enhanced by α-tocopherol.J. Biol. Chem. 1998; 273: 6080-6087Google Scholar) and their corresponding hydroxides accumulated (25Sattler W. Christison J.K. Stocker R. Cholesterylester hydroperoxide reducing activity associated with isolated high- and low-density lipoproteins.Free Radic. Biol. Med. 1995; 18: 421-429Google Scholar, 28Garner B. Waldeck A.R. Witting P.K. Rye K-A. Stocker R. Oxidation of high density lipoproteins. II. Evidence for direct reduction of HDL lipid hydroperoxides by methionine residues of apolipoproteins AI and AII.J. Biol. Chem. 1998; 273: 6088-6095Google Scholar) (data not shown). Concomitant with these changes, oxidation of apolipoproteins occurred (26Garner B. Witting P.K. Waldeck A.R. Christison J.K. Raftery M. Stocker R. Oxidation of high density lipoproteins. I. Formation of methionine sulfoxide in apolipoproteins AI and AII is an early event that correlates with lipid peroxidation and can be enhanced by α-tocopherol.J. Biol. Chem. 1998; 273: 6080-6087Google Scholar, 28Garner B. Waldeck A.R. Witting P.K. Rye K-A. Stocker R. Oxidation of high density lipoproteins. II. Evidence for direct reduction of HDL lipid hydroperoxides by methionine residues of apolipoproteins AI and AII.J. Biol. Chem. 1998; 273: 6088-6095Google Scholar), as characterized by decreasing amounts of native apoA-I and apoA-II and accumulation of new oxidized species (Fig. 1B). Three oxidized species of apoA-I were separated, two with an increase in mass of 16 Da (designated as apoA-I+16), and the third with a mass increase of 32 (apoA-I+32). MALDI-TOF analysis of these three species revealed that the mass increases were due to the oxidation of one or both Met residues 86 and 112 to MetO (Table 1). ApoA-I+16 (MetO112, eluting at ∼18.5 min in Fig. 1B) was the more prevalent of the two +16 species, perhaps suggesting increased exposure of this Met residue to the lipid hydroperoxides contained in the HDL particle. The two apoA-I+16 species accumulated before apoA-I+32 appeared. Thus, ∼10–20% of the total apoA-I was converted to apoA-I+16 species before apoA-I+32 reached detectable levels (not shown).TABLE 1ESI and MALDI-TOF analysis of native and selectively oxidized apoA-I and Met-containing peptides derived from themProteinESI MassMass PeptideResidues 73–88 (DNLEKETEGLRQE MSK)Mass PeptideResidues 108–116 (WQEEMELYR)DaapoA-I28,0811906.91283.4apoA-I+16 (Met86)28,0981923.21283.6apoA-I+16 (Met112)28,0951907.11299.5apoA-I+3228,1141923.21299.5Native or mildly oxidized (1 mM AAPH, 6 h, 37°C) HDL, was subjected to RP-HPLC, and the apolipoproteins isolated and then subjected to ESI-MS as described under Experimental Procedures. Remaining samples were lyophilized, then digested with either trypsin or AspN, and the resultant peptide fragments subjected to MALDI-TOF. ApoA-I contains three Met residues of which Met86 and Met112 become oxidized during AAPH-induced oxidation of HDL. The results shown are typical of three separate analyses using different preparations of oxHDL. Open table in a new tab Native or mildly oxidized (1 mM AAPH, 6 h, 37°C) HDL, was subjected to RP-HPLC, and the apolipoproteins isolated and then subjected to ESI-MS as described under Experimental Procedures. Remaining samples were lyophilized, then digested with either trypsin or AspN, and the resultant peptide fragments subjected to MALDI-TOF. ApoA-I contains three Met residues of which Met86 and Met112 become oxidized during AAPH-induced oxidation of HDL. The results shown are typical of three separate analyses using different preparations of oxHDL. All three apoA-II species behaved similarly with respect to oxidation, and as such will be referred to collectively throughout. ApoA-II was also converted into +16 and +32 forms by AAPH oxidation of HDL (Fig. 1B), as assessed by ESI-MS. These mass changes were the result of oxidation of one or both of the Met residues contained in the homodimer (Table 2). ApoA-II+32 co-eluted with apoA-I, so that it was only detected under relatively harsh oxidizing conditions, i.e., when all native apoA-I was oxidized (data not shown).TABLE 2ESI and MALDI-TOF analysis of native and selectively oxidized apoA-II and the Met-containing peptide derived from themProteinESI MassMass PeptideResidues 24–28 (DLMEK)DaapoA-II17,252635.3apoA-II+1617,269635.3, 651.3apoA-II+3217,289651.3Native or mildly oxidized (1 mM AAPH, 6 h, 37°C) HDL were subjected to RP-HPLC, and the apolipoproteins isolated and then subjected to ESI-MS as described under Experimental Procedures. Remaining samples were lyophilized, then digested with trypsin, and the resultant peptide fragments subjected to MALDI-TOF. ApoA-II contains a single Met residue (Met26) that becomes oxidized during AAPH-induced oxidation of HDL. The results shown are typical of three separate analyses using different preparations of oxHDL. Open table in a new tab Native or mildly oxidized (1 mM AAPH, 6 h, 37°C) HDL were subjected to RP-HPLC, and the apolipoproteins isolated and then subjected to ESI-MS as described under Experimental Procedures. Remaining samples were lyophilized, then digested with trypsin, and the resultant peptide fragments subjected to MALDI-TOF. ApoA-II contains a single Met residue (Met26) that becomes oxidized during AAPH-induced oxidation of HDL. The results shown are typical of three separate analyses using different preparations of oxHDL. To assess the rates at which the oxidized forms of apoA-I and apoA-II were formed, plasma was incubated at 37°C in the presence of AAPH, and the HDL isolated at various time points and then subjected to HPLC analysis. Oxidized forms of both apoA-I and apoA-II accumulated in a time- and AAPH-concentration-dependent manner. At low AAPH concentration, apoA-I+16 and apoA-II+16 accumulated before and at concentrations higher than apoA-I+32 (Fig. 2A), whereas these differences became smaller at higher rates of peroxyl radical generation (Fig. 2B). Both forms of apoA-I+16, i.e., MetO86 and MetO112, were formed at comparable rates and stages of oxidation, and are therefore presented together. Although it was impossible to accurately quantify apoA-I concentrations as it co-elutes with apoA-II+32 (28Garner B. Waldeck A.R. Witting P.K. Rye K-A. Stocker R. Oxidation of high density lipoproteins. II. Evidence for direct reduction of HDL lipid hydroperoxides by methionine residues of apolipoproteins AI and AII.J. Biol. Chem. 1998; 273: 6088-6095Google Scholar), it was clear from the chromatograms that formation of the oxidized species was accompanied by a decrease in native apoA-I and apoA-II (data not shown). Although AAPH is widely used as a model of controlled oxidative stress, we also wished to establish the identity of oxHDL apolipoproteins and their rate of formation during the autoxidation of whole plasma to validate the outcomes of the previous experiments. Plasma aliquots were filter-sterilized and incubated under air for up to 148 h at 37°C before HDL was isolated and analyzed by HPLC. After 24 h, both MetO86 and MetO112 were detected (Fig. 3), while apoA-I+32 was not observed until after 72 h of incubation. After 120 h, the concentrations of apoA-I+16 species detected were maximal. Subsequently, apoA-I+16 (and apoA-II+16) concentrations declined, indicating that this form of oxidized apoA-I (apoA-II) represents a transient species that bec" @default.
• W2165087479 created "2016-06-24" @default.
• W2165087479 creator A5002010316 @default.
• W2165087479 creator A5015511874 @default.
• W2165087479 creator A5033253669 @default.
• W2165087479 creator A5040526592 @default.
• W2165087479 creator A5041043738 @default.
• W2165087479 creator A5044450748 @default.
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• W2165087479 date "2003-02-01" @default.
• W2165087479 modified "2023-10-15" @default.
• W2165087479 title "Characterization of specifically oxidized apolipoproteins in mildly oxidized high density lipoprotein" @default.
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